International Journal of Systematic and Evolutionary Microbiology (2002), 52, 913–920 DOI: 10.1099/ijs.0.02034-0

Thioalkalimicrobium cyclicum sp. nov. and Thioalkalivibrio jannaschii sp. nov., novel of haloalkaliphilic, obligately chemolithoautotrophic -oxidizing from hypersaline alkaline Mono Lake (California)

1 Institute of Microbiology, Dimitry Yu. Sorokin,1,3 Vladimir M. Gorlenko,1 Tat’yana P. Tourova,1 Russian Academy of 2 2 3 Science, Prospect 60-let Alexandre I. Tsapin, Kenneth H. Nealson and Gijs J. Kuenen Octyabrya 7/2, 117811 Moscow, Russia

2 Author for correspondence: Dimitry Yu. Sorokin. Tel: j7 95 1350109. Fax: j7 95 1356530. Jet Propulsion e-mail: soroc!inmi.da.ru Laboratory/California Institute of Technology, Pasadena, CA, USA Two strains of haloalkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria 3 Kluyver Institute of were isolated from the oxygen-sulfide interface water layer of stratified Biotechnology, TU Delft, T Julianalaan 67, 2628 BC alkaline and saline Mono Lake, California, USA. Strain ALM 1 was a dominant Delft, The Netherlands species in enrichment on moderate-saline, carbonate-buffered medium (06M total NaM, pH 10) with thiosulfate as an energy source and nitrate as a source. Cells of ALM 1T are open ring-shaped and are non-motile. It has a high growth rate and activity of thiosulfate and sulfide oxidation and very low sulfur-oxidizing activity. Genetic comparison and phylogenetic analysis suggested that ALM 1T (l DSM 14477T l JCM 11371T) represents a new species of the genus Thioalkalimicrobium in the γ-, for which the name Thioalkalimicrobium cyclicum sp. nov. is proposed. Another Mono Lake isolate, strain ALM 2T, dominated in enrichment on a medium containing 2 M total NaM (pH 10). It is a motile vibrio which tolerates up to 4 M NaM and produces a membrane-bound yellow pigment. Phylogenetic analysis placed ALM 2T as a member of genus Thioalkalivibrio in the γ-Proteobacteria, although its DNA hybridization with the representative strains of this genus was only about 30%. On the basis of genetic and phenotypic properties, strain ALM 2T (l DSM 14478T l JCM 11372T) is proposed as Thioalkalivibrio jannaschii sp. nov.

Keywords: haloalkaliphilic sulfur-oxidizers, soda lakes, Mono Lake, Thioalkalimicrobium, Thioalkalivibrio

INTRODUCTION alkaline pH are represented by saline soda lakes, and recent intensive microbiological investigation of these Colourless sulfur-oxidizing bacteria represent an im- habitats has revealed the presence of alkaliphilic portant part of microbial community in natural and bacteria belonging to various physiological groups some industrial habitats. Until recently, known sulfur- (Jones et al., 1998; Zavarzin et al., 1999). In particular, oxidizing bacteria included neutrophilic and acido- it became clear that there exists a third, halo- philic species with possible pH range for stable growth alkaliphilic, group of sulfur-oxidizing bacteria, which between 2 and 8 (Kelly, 1989; Kuenen et al., 1992). includes both heterotrophic (Sorokin et al., 1996) and Natural environments with high alkalinity and stable obligately autotrophic (Sorokin et al., 2000b) repre-

...... sentatives. Overall, more than 80 strains of alkaliphilic, Published online ahead of print on 14 December 2001 as DOI obligately autotrophic, sulfur-oxidizing strains have 10.1099/ijs.0.02034-0. been isolated and characterized (Sorokin et al., 2001a) The GenBank accession numbers for the 16S rDNA sequence of strains ALM from the volcanic soda lakes in Kenya and from the 1T and ALM 2T are AF329082 and AF329083, respectively. shallow soda lakes in dry steppes of the south-east

02034 # 2002 IUMS Printed in Great Britain 913 D. Yu. Sorokin and others

Siberia and Mongolia. In general, these bacteria differ experiments. The pH influence on the activity of thiosulfate from the well-known neutrophilic species by their oxidation was tested with an oxygen electrode as described ability to grow optimally at pH " 9 and up to 10n5–10n6 previously (Sorokin et al., 2001a) using 0n1 M HEPESj in media strongly buffered by a sodium bicarbonate\ 0n6 M NaCl for the pH range 6–8 and NaHCO$\Na#CO$ carbonate mixture. All these bacteria have been buffer for higher pH values. All buffers contained 50 mM KCl. The influence of salt concentration on the activity of assigned into two new genera Thioalkalimicrobium and thiosulfate oxidation was investigated using soda buffer, pH Thioalkalivibrio in the γ-Proteobacteria (Sorokin et al., 10, with total Na+ concentration from 0n3to4M. 2001a). The two genera differ from each other in many aspects of the growth kinetics, metabolic activity and Total DNA analysis. The isolation of the DNA and sub- sequent determination of the GjC content of the DNA and genetics. In general, the genus Thioalkalimicrobium the DNA–DNA hybridization were performed according to includes fast-growing species with high activity of standard procedures (Marmur, 1961; De Ley et al., 1970). thiosulfate and sulfide oxidation but relatively low salt tolerance. Most of the strains were obtained from the Amplification and sequencing of 16S rRNA genes. For amplification and sequencing of 16S rRNA genes, the DNA low-mineralized steppe soda lakes. The genus is a was obtained by standard phenol\chloroform extraction. member of the Thiomicrospira cluster. In contrast, the The 16S rRNA genes were selectively amplified using primers genus Thioalkalivibrio is represented by the slow- 5h-AGAGTTTGATCCTGGCTCAG-3h (forward) and 5h- growing but more salt-tolerant organisms isolated TACGGTTACCT-TGTTACGACTT-3h (reverse). PCR mostly from the highly concentrated Kenyan soda products were cloned, and transformed using InvitroGene lakes. Some of the isolates were even capable of growth kit. Sequencing had been done by MWG Biotech, Inc (High in saturated soda brines. The genus Thioalkalivibrio is Point North Carolina) with Licor machine using custom- related to sulfur purple bacteria of the genus designed primers to sequence samples to an accuracy of Ectothiorhodospira (Tru$ per & Schlegel, 1964). This " 99%. A combination of both available sequencing genus also includes several strains capable of growth reactions which include dye primer on the Licor Long Read IR 4200 sequencers and dye terminator on the ABI3700 with thiocyanate as electron donor and nitrogen source capillary sequencers were used. Dye primer chemistry (Sorokin et al., 2001b) which form two new Thio- provided data read lengths from 750 to over 1100 bases, alkalivibrio species (Sorokin et al., 2002). In while the dye-terminator reaction was used to confirm base this paper we describe two new species of halo- calls and provide gap closure with read lengths ranging from alkaliphilic, obligately chemolithoautotrophic, sulfur- 500 to 750 bases. Nearly complete 16S rDNA gene sequences oxidizing bacteria isolated from the stratified, alkaline were obtained for the Mono Lake isolates ALM 1T and and saline Mono Lake in California, USA. ALM 2T (1450–1470 nucleotides). 16S rDNA sequence analysis. The sequences were aligned METHODS manually with sequences obtained from the database of small-subunit rRNAs in EMBL. The sequences were Sampling. Water samples were collected in July 1999 from compared with those of the members of the Proteobacteria. the sulfide-oxygen interface layer (depth 19–25 m) at the Regions that were not sequenced in one or more reference deepest point (48 m) of Mono Lake, California, using 5 l organism were omitted from the analyses. Pairwise evol- sampling bottles. The mean pH and total salinity values of " utionary distances (expressed as estimated changes per 100 the Mono Lake water were 9n8 and 80 g l− , respectively. The nucleotides) were computed by using the Jukes & Cantor HS− concentration decreased upwards from several hundred method. A resulting phylogenetic tree was constructed by micromolar in the anaerobic layer to several micromolar in the neighbour-joining method (Saitou & Nei, 1987) with the upper part of the interface layer. The samples were bootstrap analysis of 100 trees using programs of the brought into a laboratory within 5 h of collection and kept  package (Van de Peer & De Wachter, 1994). at 4 mC until use. Bootstrap analysis (100 replications) was used to validate Media and growth conditions. Enrichment for and cul- the reproducibility of the branching pattern of the trees. tivation of aerobic alkaliphilic sulfur bacteria was per- Electron microscopy. For total preparations, cells were formed using a mineral medium strongly buffered by washed and resuspended in 0n5–1 M neutral NaCl solution, + NaHCO$\Na#CO$ (0n6–4 M total Na ) at pH 10–10n1, as pre-fixed (2 h) and then fixed (10 h) at 4 mCin0n1% and described previously (Sorokin et al., 2001a). Thiosulfate 2n5% (v\v, final) glutaralaldehyde solutions, respectively, (40–80 mM) served as the energy source and nitrate (5 mM containing the same amount NaCl, and then positively as KNO$) as the nitrogen source. Solid alkaline media with stained with 1% (w\v) uranyl acetate. For ultrathin final salt concentrations 0n6 and 2 M total Na+ were prepared sectioning cells after fixation were postfixed in 1% (w\v) by 1:1 mixing of 4% agar and double-strength mineral base OsO% solution containing 0n6–2 M NaCl for 10 h at 4 mC. at 50 mC. Enrichments for denitrifying sulfur bacteria were Then the cells were washed, dehydrated and embedded into performed in 100 ml serum bottles with butyl rubber the resin. Thin sections were stained with 1% solutions stoppers filled with 50 ml of alkaline base with 20 mM (w\v) of uranyl acetate and lead citrate. thiosulfate and 20 mM nitrate. Anaerobiosis was achieved Chemical analysis. Thiosulfate in batch cultures and in by five cycles of evacuation-flushing with argon. experiments with washed cells was determined by the Activity tests. The activity tests were performed with washed iodimetric titration after acidification of the samples by cells obtained from batch cultures grown at pH 10 with acetic acid to pH 5. Sulfide was measured colorimetrically thiosulfate. Cells were harvested by centrifugation, washed (Tru$ per & Schlegel, 1964) after precipitation with zinc twice with soda buffer and resuspended in the same buffer at acetate (1%, w\v, final). Elemental sulfur was extracted " 20 mg protein ml− and kept on ice. This concentrated from the cell pellets with acetone overnight and was assayed suspension was diluted 100–1000 times immediately before by a cyanolytic procedure (So$ rbo, 1957). The biomass

914 International Journal of Systematic and Evolutionary Microbiology 52 Haloalkaliphilic sulfur-oxidizing bacteria protein was determined by the Lowry method, interfering (a) sulfur compounds being removed either by several washings of the cell pellet with 0n6 M NaCl (thiosulfate) or by a double acetone extraction (elemental sulfur). The pigment from the cells of strain ALM 2 was extracted with a methanol\acetone (1:1) mixture. Its absorption spectrum was recorded on the UV-Visible diode-array Hewlett Packard HP 8453 spectro- photometer.

RESULTS AND DISCUSSION Enrichment and isolation of pure cultures Two different positive enrichment cultures from the interface water layer of Mono Lake were obtained using alkaline mineral medium with thiosulfate and different salt content. In low-salt enrichment (0n6M Na+) a phenotype with coccoid, non-motile cells was $ % " seen. Tentative enumeration showed 10 –10 cells ml− . + In high-salt enrichment (2n5M Na ) slightly curved motile rods dominanted, and the cultures turned yellowish after complete consumption of thiosulfate and intermediately produced elemental sulfur. Plating of the cultures onto solid media with corresponding (b) salt concentrations resulted in isolation of two different pure cultures of aerobic sulfur-oxidizing bacteria, T + T + strains ALM 1 (0n6MNa ) and ALM 2 (2 M Na ).

Morphology The colonies of strain ALM 1T were up to 3 mm in diameter, reddish, transparent, without sulfur depo- sition. Under the light microscope the cells looked like tiny, non-motile, irregular spheres, often aggregated. The electron microscopy demonstrated that the cells of ALM 1T were shaped as the open rings (Fig. 1a, b) with a diameter 0n5–0n8 µm and cell width 0n3–0n4 µm. Many cells contained multiple carboxysome-like structures (Fig. 1a). The colonies of strain ALM 2T were up to 4 mm in diameter, yellowish, with sulfur deposition. T ALM 2 cells are curved rods, 0n3–0n4i1–2 µm, motile by a single polar flagellum (Fig. 2a). Cells often contained intracellular sulfur globules (Fig. 2b, c) apparently encapsulated via invagination of the cell T membrane (Fig. 2d). The biomass of ALM 2 was ...... yellow-coloured due to membrane-associated pigment. Fig. 1. Morphology of alkaliphilic sulfur bacterium strain ALM T Methanol\acetone extract of the cell membranes had 1 from Mono Lake (electron photomicrographs of thin sections; bars, 0n5 µm) grown with thiosulfate at pH 10 and + absorption maxima at 405, 430 (main) and 450 nm. 0n6MNa . C, carboxysome-like structures. The specific pigment content in the biomass increased about twofold when salt content of the growth medium increased from 0 6 to 2–2 5 M total Na+, remaining at n n T T constant level upon further increase of salinity to quence similarity to type strains AL 3 and AL 7 T 3–4 M Na+. (96n8–98n5%). Strain ALM 2 is more distantly related but definitely belongs to a phylogenetic cluster of the genus Thioalkalivibrio (92n7–96n6% sequence simi- Phylogeny larity) (Fig. 3). Phylogenetic analysis of nearly complete 16S rDNA T T gene sequences of strains ALM 1 and ALM 2 Genetic analysis demonstrated their close relationship to two recently described genera of alkaliphilic sulfur bacteria in the γ- The results of total DNA analysis of the Mono Lake T Proteobacteria. Strain ALM 1 is a member of the isolates are given in Table 1. The GjC content in genus Thioalkalimicrobium with a high level of se- DNA of both strains fitted well into the range typical http://ijs.sgmjournals.org 915 D. Yu. Sorokin and others

(a) (b)

(c)

(d)

...... Fig. 2. Morphology of alkaliphilic sulfur bacterium strain ALM 2T from the Mono Lake (electron photomicrographs) grown with thiosulfate at pH 10 and 2 M Na+. (a) Total preparation (bar, 1 µm); (b)–(d), thin sections (bar, 1 µm). S, Intracellular sulfur globules; M, circular membrane envaginates.

for the corresponding genera of previously described served. Like other sulfur-oxidizing strains isolated haloalkaliphilic sulfur bacteria. Comparison of total previously from various soda lakes, the Mono Lake DNA of strain ALM 1T and the type strains of the isolates were obligately alkaliphilic. Growth in batch + genus Thioalkalimicrobium confirmed the results of culture with thiosulfate at 0n6MNa salt content was phylogenetic analysis, in that this strain, despite its possible within the pH range 7n5–10n5. The thiosulfate- peculiar morphology, is a member of the genus dependent O# consumption by washed cells of strain Thioalkalimicrobium. It had relatively high hybridi- ALM 1T and ALM 2T was observed within the pH zation to rod-shaped type strain Thioalkalimicrobium range 6n5–11 and 7n0–11n2, respectively, with an opt- T aerophilum AL 3 but it was still less than is usual imum at pH 9n5 for both organisms (Fig. 4). The salt T between strains of the same species (! 70%; tolerance of strain ALM 1 was in the moderate range Stackebrandt & Goebel, 1994). In contrast, strain while ALM 2T belongs to an extremely tolerant type, ALM 2T showed a close phenotypic similarity to the being able to function within a very broad salinity + members of genus Thioalkalivibrio, in particular with range from 0n4 to 4 M total Na (Fig. 5).The difference the yellow coloured, extremely salt-tolerant subgroup, between the strains was more evident with growing but displayed a very low level of DNA hybridization cultures (measured by the rate of thiosulfate con- (! 32%) with the representative strains of Thio- sumption, Fig. 5a) as compared to activity of the alkalivibrio. Nevertheless, with clear phenotypic and resting cells (Fig. 5b). Strain ALM 1T never formed phylogenetic indications, strain ALM 2T should be sulfur as an intermediate during growth with thio- considered as a member of the genus Thioalkalivibrio. sulfate, while prolific sulfur production was observed in ALM 2T cultures at salt concentration less than 2 M + Physiological properties total Na . Both Mono Lake isolates belong to obligately chemo- The respiratory profiles of both strains (Table 2) are lithoautotrophic sulfur-oxidizing bacteria. Growth consistent with the patterns of representatives of the with hydrogen as electron donor and denitrification genera Thioalkalimicrobium and Thioalkalivibrio.Asis (thiosulfatejnitrate, nitrite or N#O) were not ob- typical for members of the genus Thioalkalimicrobium

916 International Journal of Systematic and Evolutionary Microbiology 52 Haloalkaliphilic sulfur-oxidizing bacteria

...... Fig. 3. Phylogenetic tree showing the relationships of the Mono Lake strains of alkaliphilic sulfur-oxidizing bacteria in the γ-Proteobacteria. The numbers on the branches refer to bootstrap values; only values above 90% are shown. The bar represents 5% sequence divergence.

Table 1. DNA–DNA hybridization between Mono Lake isolates and members of genera Thioalkalimicrobium and Thioalkalivibrio ...... Thioalkalimicrobium: group 1 – Thioalkalimicrobium aerophilum AL 3T; group 2–Thioalkalimicrobium sibericum AL 7T. Thioalkalivibrio: group 1 – yellow-coloured, extremely salt-tolerant strains from Mongolian, Kenyan and Egyptian soda lakes; group 2 – low-salt tolerant, colourless strains from Kenyan and Siberian soda lakes. Non-specific background values for DNA hybridization (Escherichia coli) was not higher than 5%.

Strain DNA GjC content DNA–DNA hybridization (%) (mol%) Thioalkalimicrobium Thioalkalivibrio

Group 1 Group 2 Group 1 (8 strains) Group 2 (6 strains)

ALM 1T 49n66030   ALM 2T 63n7   25–32 15–20

, No data.

(Sorokin et al., 2001a), strain ALM 1T oxidized Mono Lake belongs to a comparatively rare type of thiosulfate and sulfide, less actively polysulfide and stratified alkaline and highly saline lakes. In this lake, tetrathionate and had no activity with elemental sulfur. a hypersaline, anaerobic, sulfide-containing water Strain ALM 2T oxidized all five sulfur compounds with body is overlaid by less saline, aerobic waters. The more or less equal but relatively low activity, which result is a rather stable oxic\anoxic interface that has is a characteristic of the members of genus Thio- been shown to act as an active aerobic chemo- alkalivibrio (Sorokin et al., 2001a). Addition of sulfite lithotrophic ‘filter’ for reduced inorganic compounds, did not stimulate oxygen consumption in the cell such as , methane and sulfide (Joye et al., suspensions of the new isolates. 1999; Ward et al., 2000). Given the high sulfate http://ijs.sgmjournals.org 917 D. Yu. Sorokin and others

Table 2. Respiration rate of washed cells ...... ALM 1T and ALM 2T were grown with thiosulfate at pH 10 + −" and 0n6MNa . Values are given as µmol O# (mg protein) " min− (means of 2–3 independent measurements); endogenous rates were subtracted.

Substrate Respiration rate

ALM 1T ALM 2T

#− Thiosulfate (S#O$ )2n50 0n60 Sulfide (HS−)3n04 0n70 #− Polysulfide (S' )1n80 0n66 ...... Sulfur (S))0n00 0n52 #− Fig. 4. Influence of pH on the activity of thiosulfate-dependent Tetrathionate (S%O' )0n42 0n33 oxygen consumption by washed cells of the Mono Lake isolates ALM 1T ($) and ALM 2T (#). All buffers contained 0n6 M total + Na .pH6n5–8, HEPESjNaCl; pH 8–11n2, NaHCO3/Na2CO3. Means from the two independent experiments. cultivable types of obligately chemolithoautotrophic, aerobic, haloalkaliphilic sulfur bacteria inhabit this lake. One type (represented by strain ALM 1T), enriched at moderate salt concentration, probably corresponds to a population adapted to the surface aerobic waters of the Mono Lake mixolimnion. The other type (represented by strain ALM 2T), enriched at extremely high salt concentrations, functions opti- mally at 1–3 M Na+, which encompasses the salt + content of the monimolimnion (1n4MNa ) but also would allow such bacteria to survive a local increase of salinity. Such conditions may occur in the shallow evaporative pools temporarily separated from the main lake water body. Unlike many other freshwater and saline stratified shallow lakes (where sufficient light penetrates to a sulfide-containing layer), in Mono Lake the anaerobic sulfide-oxidizing phototrophs do not build up a sub- stantial population (V. Gorlenko, unpublished). The reasons are not quite clear. It could be presence of unusually high concentrations of toxic oxyanions, such #− #− AsO% and SeO$ (Stolz & Oremland, 1999) or light interception by the abundant mixolimnion population of the unicellular algae ‘Picocystis salinarium’, which was recently discovered in the Mono Lake ecosys- tem (http:\\www.monobasinresearch.org\research\ microbial). Whatever the reason, in the absence of active population of anaerobic phototrophs it might be speculated that the aerobic, haloalkaliphilic, sulfur- ...... oxidizing bacteria play a key role in the oxidation of Fig. 5. Influence of salt concentration (NaHCO /Na CO /NaCl) 3 2 3 sulfide coming up from the anaerobic layer in Mono on growth and activity of thiosulfate oxidation by washed cells of the Mono Lake isolates at pH 10. (a) Amount of thiosulfate Lake. consumed during 24 and 47 h growth in batch cultures of the On the basis of physiological and genetic properties, strains ALM 1T and ALM 2T, respectively; (b) activity of thiosulfate-dependent respiration. $, ALM 1T ; #, ATM 2T. the Mono Lake isolates were clearly related to two Means from the two independent experiments. different groups of previously described haloalka- liphilic sulfur-oxidizing bacteria isolated from the Kenyan and Siberian soda lakes (Sorokin et al., 2001a). Strain ALM 1T, being physiologically very similar to concentration (130 mM) (Oremland & Miller, 1993), representatives of the genus Thioalkalimicrobium, the presence of sulfur-oxidizing bacteria in the redox differs from the known species of this genus by its interface of Mono Lake was suspected as well. Our morphology. Phylogenetically strain ALM1T is closely investigation demonstrated that at least two different related to members of the genus Thioalkalimicrobium

918 International Journal of Systematic and Evolutionary Microbiology 52 Haloalkaliphilic sulfur-oxidizing bacteria but since its DNA similarity with described species was maxima at 405, 430 (main) and 450 nm. Obligately less than 70%, this strain is proposed as a new species, chemolithoautotrophic, haloalkaliphilic bacterium. Thioalkalimicrobium cyclicum. Strain ALM 2T, despite Tolerates up to 4 M Na+. Oxidizes sulfide, thiosulfate, its very low DNA hybridization with the representative polysulfide, sulfur and tetrathionate. GjC content in strains, phenotypically clearly resembles the extremely DNA is 63n7mol%(Tm). Isolated from the O#-sulfide salt-tolerant members of the genus Thioalkalivibrio.In interface layer of alkaline Mono Lake (California). view of the results of phylogenetic analysis, this isolate Other properties as for the genus. The type strain is T T T should be affiliated with the genus Thioalkalivibrio and ALM 2 (l DSM 14478 l JCM 11372 ). is proposed as a new species Thioalkalivibrio jannaschii. We have also found this type of sulfur-oxidizing double extremophiles dominating in highly saline alkaline ACKNOWLEDGEMENTS lakes in Mongolia, Kenya and Wadi an Natrun in Egypt (Sorokin et al., 2001a; our unpublished results). This work was financially supported by The Netherlands It is interesting to mention that these sulfur-oxidizing Organization for Scientific Research (NWO Project bacteria probably represent the only type of aerobic 047.006.018) and RFBR (99-04-48707 and 01-04-48782). chemolithoautotrophs capable to grow in saturated Part of this work was performed at the Jet Propulsion alkaline brines. Other types of alkaliphilic chemo- Laboratory\California Institute of Technology, with sup- port from DRDF grant 100656-00888. We thank Diane lithoautotrophs (methane-, hydrogen-, ammonia- and Engler for technical assistance. nitrite-oxidizing), although being detected in and isolated from the extremely saline soda lakes, are able to grow only at salinity below 1 M Na+ (Ward et al., 2000; Sorokin et al., 1998, 2000a, c, 2001c). REFERENCES De Ley, J., Caffon, H. & Reinaerts, A. (1970). The quantitative measurements of DNA hybridization from renaturation rates. Eur J Description of Thioalkalimicrobium cyclicum sp. nov. Biochem 12, 133–140. Thioalkalimicrobium cyclicum (cyc.li cum Gr. n. cyclus Jones, B. E., Grant, W. D., Duckworth, A. W. & Owenson, G. G. h (1998). Extremophiles 2 circle; M.L. n. cyclicum circle-like). Microbial diversity of soda lakes. , 191–200. Joye, S. B., Conell, T. L., Miller, L. G., Oremland, R. S. & Jellison, Cells are mostly in a form of open ring with a diameter R. S. (1999). Oxidation of ammonia and methane in an alkaline, saline 0n5–0n8 and cell width 0n3–0n4 µm. 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